The invention relates to proteolytic processing of the xcex2-amyloid precursor protein, and more particularly to assays for determining factors that affect such processing.
A number of important neurological diseases including Alzheimer""s disease (AD), cerebral amyloid angiopathy (CAA), and prion-mediated diseases are characterized by the deposition of aggregated proteins, referred to as amyloid, in the central nervous system (CNS) (for reviews, see Glenner et al. (1989) J Neurol. Sci. 94:1-28; Haan et al. (1990) Clin. Neurol. Neurosurg. 92(4):305-310. These highly insoluble aggregates are composed of nonbranching, fibrillar proteins with the common characteristic of a xcex2-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur (1989) Acta Neuropathol. 78:329-331; Kawai et al. (1993) Brain Res. 623:142-6; Martin et al. (1994) Am. J. Pathol. 145:1348-1381; Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996) J. Neurosci. 16:5795-5811). AD studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996) Nat. Med. 2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy (1997) Trends Neurosci. 20:154-9).
AD and CAA share biochemical and neuropathological markers, but differ somewhat in the extent and location of amyloid deposits as well as in the symptoms exhibited by affected individuals. The neurodegenerative process of AD, the most common cause of progressive intellectual failure in aged humans, is characterized by the progressive and irreversible deafferentation of the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaque and tangle formation (for a review see Terry et al. (1994) xe2x80x9cStructural alteration in Alzheimer""s disease.xe2x80x9d In: Alzheimer""s disease (Terry et al. eds.), pp. 179-196. Raven Press, New York). Dystrophic neurites, as well as reactive astrocytes and microglia, are associated with these amyloid-associated neurite plaques. Although, the neuritic population in any given plaque is mixed, the plaques generally are composed of spherical neurites that contain synaptic proteins, APP (type I), and fusiform neurites containing cytoskeletal proteins and paired helical filaments (PHF; type II).
CAA patients display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage. Cerebral parenchymal hemorrhage is the result of extensive amyloid deposition within cerebral vessels (Hardy (1997) Trends Neurosci. 20:154-9; Haan et al. (1990) Clin. Neurol. Neurosurg. 92:305-10; Terry et al., supra; Vinters (1987) Stroke 18:211-24; Itoh et al. (1993) J. Neurological Sci. 1 16:135-41; Yamada et al. (1993) J Neurol. Neurosurg. Psychiatry 56:543-7; Greenberg et al. (1993) Neurology 43:2073-9; Levy et al. (1990) Science 248:1124-6). In some familial CAA cases, dementia was noted before the onset of hemorrhages, suggesting the possibility that cerebrovascular amyloid deposits may also interfere with cognitive functions.
In both AD and CAA, the main amyloid component is the amyloid protein (Axcex2). The Axcex2 peptide, which is generated from the amyloid precursor protein (APP) by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, Axcex21-40 and Axcex21-42, are produced by alternative carboxy-terminal truncation of APP (Selkoe et al.(1988) Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409). Axcex21-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy (1997), supra; Haan et al. (1990), supra; Terry et al., supra; Vinters (1987), supra; Itoh et al. (1993), supra; Yamada et al. (1993), supra; Greenberg et al. (1993), supra; Levy et al. (1990), supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.
The formation of Axcex2 is considered to be a key pathogenic process in Alzheimer""s disease and related neurodegenerative disorders (reviewed by Selkoe in Nature Suppl.399: A23, 1999). The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the AD-associated, and CAA-associated neurodegenerative processes are not well-defined. However, evidence indicates that dysregulated expression and/or processing of APP gene products or derivatives of these gene products derivatives are involved in the pathophysiological process leading to neurodegeneration and plaque formation. For example, missense mutations in APP are tightly linked to autosomal dominant forms of AD (Hardy (1994) Clin. Geriatr. Med. 10:239-247; Mann et al. (1992) Neurodegeneration 1:201-215). The role of APP in neurodegenerative disease is further implicated by the observation that persons with Down""s syndrome who carry an additional copy of the human APP (hAPP) gene on their third chromosome 21 show an overexpression of hAPP (Goodison et al. (1993) J. Neuropathol. Exp. Neurol. 52:192-198; Oyama et al. (1994) J. Neurochem. 62:1062-1066) as well as a prominent tendency to develop AD-type pathology early in life (Wisniewski et al. (1985) Ann. Neurol. 17:278-282). Mutations in Axcex2 are linked to CAA associated with hereditary cerebral hemorrhage with amyloidosis (Dutch (HCHWA-D)(Levy et al. (1990), supra), in which amyloid deposits preferentially occur in the cerebrovascular wall with some occurrence of diffuse plaques (Maat-Schieman et al. (1994) Acta Neuropathol. 88:371-8; Wattendorff et al. (1995) J. Neurol. Neurosurg. Psychiatry 58:699-705). A number of hAPP point mutations that are tightly associated with the development of familial AD encode amino acid changes close to either side of the Axcex2 peptide (for a review, see, e.g., Lannfelt et al. (1994) Biochem. Soc Trans. 22:176-179; Clark et al. (1993) Arch. Neurol. 50:1164-1172). Finally, in vitro studies indicate that aggregated Axcex2 can induce neurodegeneration (see, e.g., Pike et al. (1995) J. Neurochem. 64:253-265).
APP is a glycosylated, single-membrane-spanning protein expressed in a wide variety of cells in many mammalian tissues. Examples of specific isotypes of APP which are currently known to exist in humans are the 695-amino acid polypeptide described by Kang et al. (1987) Nature 325:733-736, which is designated as the xe2x80x9cnormalxe2x80x9d APP. A 751-amino acid polypeptide has been described by Ponte et al. (1988) Nature 331:525-527 and Tanzi et al. (1988) Nature 331:528-530. A 770-amino acid isotype of APP is described in Kitaguchi et al. (1988) Nature 331 :530-532. A number of specific variants of APP have also been described having point mutations which can differ in both position and phenotype. A general review of such mutations is provided in Hardy (1992) Nature Genet. 1:233-234. A mutation of particular interest is designated the xe2x80x9cSwedishxe2x80x9d mutation where the normal Lys-Met residues at positions 595 and 596 are replaced by Asn-Leu. This mutation is located directly upstream of the normal xcex2-secretase cleavage site of APP, which occurs between residues 596 and 597 of the 695 isotype.
APP is post-translationally processed by several proteolytic pathways resulting in the secretion of various fragments or intracellular fragmentation and degradation. F. Checler, J. Neurochem. 65:1431-1444 (1995). The combined activity of xcex2-secretase and xcex3-secretase on APP releases an intact xcex2-amyloid peptide (Axcex2), which is a major constituent of amyloid plaques. Axcex2 is an approximately 43 amino acid peptide which comprises residues 597-640 of the 695 amino acid isotype of APP. Internal cleavage of APP by xcex1-secretase inhibits the release of the full-length Axcex2 peptide. Although the extent of pathogenic involvement of the secretases in AD progression is not fully elucidated, these proteolytic events are known to either promote or inhibit Axcex2 formation, and thus are thought to be good therapeutic candidates for AD.
Although a number of assays have been developed to examine secretase activity, each of these has limitations. Available cell-free assays which typically utilize synthetic substrates are not entirely reflective of the in vivo situation. Also, conditions of preparation and performing the cell-free assays have not been designed to reflect the in vivo state. Whole cell assays, although they accurately reflect physiological states of enzyme activity, are more difficult because the agents affecting enzymatic activity must be permeable to the cell as well as the subcellular compartments.
Numerous reports have been made describing the isolation and identification of putative xcex3-secretases (reviewed by Evin et al. in Amyloid 1: 263, 1994). It has been proposed that PS1 is xcex3-secretase (Wolfe et al. Nature 398: 513, 1999). The evidence supporting this proposal is indirect but has nonetheless prompted considerable discussion (Wolfe et al. Biochem. 38: 11223, 1999; Annaert and De Strooper TINS 22: 439, 1999). However, none of these putative xcex3-secretases has been definitively proven to be the authentic activity capable of producing Axcex2. The ability to produce Axcex2 in vitro using a solubilized mammalian cell extract allows for purification and definitive identification of xcex3-secretase activity. Moreover, this assay has the advantage that (1) it uses the native substrate derived from xcex2-amyloid precursor protein, APP, and not synthetic or chimeric APP substrates and (2) the xcex3-secretase activity is monitored by following the production of authentic Axcex2 protein. In addition, two major Axcex2 isoforms are generated by xcex3-secretase action, Axcex240 and Axcex242, representing 40 and 42 amino acid long proteins, respectively. It has been debated whether a single enzymatic activity is responsible for the generation of all Axcex2 isoforms or whether distinct xcex3-secretases exist, one generating each Axcex2 isoform. With this solubilized system for xcex3-secretase activity this issue can be resolved. The generation of specific Axcex2 isoforms can be assessed and the enzymatic activity for each purified, if multiple activities exist. If only one enzyme produces the Axcex2 isoforms, this will be apparent in the purification process. Knowledge of single versus multiple xcex3-secretases is important with regard to development of inhibitors of xcex3-secretase and Axcex2 formation. In particular, it may be advantageous to selectively inhibit Axcex242 as this has been shown to be more pathogenic by a number of criteria (reviewed by Selkoe in J. Biol. Chem. 271: 18295, 1996; Sotrey and Cappai in Neuropath. and Appl. Neurol. 25: 81, 1999). Furthermore, because the xcex3-secretase cleavage site on APP appears to be located in the transmembrane domain, the role of lipids or a membrane-like milieu can also be assessed with this assay system.
It is evident from a number of reports that presenilin proteins (PS1 and PS2) are involved in xcex3-secretase processing of Axcex2. For example, cells from PS1 knock out mice display a reduced level of Axcex2 protein and a concomitant increase in the immediate precursor to Axcex2, the carboxyl-terminal 99 residue domain of APP (De Strooper et al. Nature 391: 387, 1998). PS2 knock out mice do not appear to significantly influence xcex3-secretase processing to Axcex2 (Herreman et al. Proc. Natl. Acad. Sci. USA 96: 11872, 1999) although other reports suggest a role for PS2 in Axcex2 formation (Jacobsen et al. J. Biol. Chem. 274: 35233, 1999; Steiner et al. J. Biol. Chem. 274: 28669, 1999). More recent evidence in fact suggests that PS1, although necessary for xcex3-secretase activity, is not in fact xcex3-secretase (Octave et al., J. Biol. Chem 275: 1525-1528).
There is thus a need in the art for a system to identify the molecules responsible for xcex3-secretase activity. There is also a need for an efficient and reproducible method of identifying inhibitors/modulators of xcex3-secretase activity that affect APP processing. There is especially a need for an assay that more accurately reflects in vivo activity of this enzymatic activity.
The present invention provides a solubilized xcex3-secretase system and a cell-free assay for identifying modulators of the APP processing enzyme xcex3-secretase. The method of the invention utilizes either a membrane source of both APP and xcex3-secretase or individual membrane sources for APP and secretase to provide a solubilized xcex3-secretase activity and to determine factors that may enhance or decrease enzymatic activity affecting Axcex2 peptide production. The cell membranes used in the assay may be from cells expressing an endogenous APP or, preferably, cells expressing a recombinant human APP. The APP may be full-length or a fragment capable of being proteolytically cleaved by xcex3-secretase, e.g., CT99. In addition, the APP expressed in the cells may have one or more mutation, such as a point mutation, small deletion, etc.
In one embodiment, the invention provides a method for producing solubilized xcex3-secretase activity. The isolated solubilized xcex3-secretase activity can be used for a variety of purposes including the generation of Axcex2 and the identification and isolation of molecules(s) responsible for the xcex3-secretase activity. Identification and characterization of the molecules responsible for xcex3-secretase activity will allow the rational design of inhibitors which act directly on xcex3-secretase to prevent Axcex2 formation. The solubilized xcex3-secretase activity can be isolated with APP or an APP proteolytic product, or it may be reconstituted as a mixture between isolated xcex3-secretase activity and APP or an APP proteolytic product.
In another embodiment, crude membrane preparations having xcex3-secretase activity are provided. These membrane preparations are composed of at least the xcex3-secretase activity, but may also have numerous other membrane-bound cellular products. These membrane preparations are particularly useful in determining the potential effect of agents that affect xcex3-secretase activity on other membrane molecules.
In yet another embodiment, the invention provides an assay for identifying compounds which modulate xcex3-secretase activity. The assay method of the invention includes the steps of 1) obtaining cell membranes from cells expressing endogenous and/or exogenous APP or an APP proteolytic product (e.g., recombinant APP), 2) removing background xcex3-secretase products of APP (e.g., Axcex2, p3 and/or xcex3CTF) present in the cells at the time of preparation; 3) incubating the membrane preparations with an agent that potentially modifies xcex3-secretase activity; and 4) detecting the proteolytic products of xcex3-secretase, e.g., xcex3CTF, p3 and/or Axcex2. Detection may be accomplished using an antibody that selectively recognizes the xcex3-secretase products, or by other methods such as isolation and characterization of the xcex3-secretase products. The present assays can detect very small increases in proteolytic product, with an assay being sensitive within a range of 0.5 to 4 ng/ml product for an ELISA assay and within a range of 0.002-0.05 ng/ml using Western Blot analysis of the product.
In a particular embodiment, the membranes used for isolation of xcex3-secretase activity are obtained from cells that express a gene altered from a normal, endogenous gene. For example, the cells may express an APP altered to be more or less vulnerable to secretase activity, e.g. mutations to increase xcex3-secretase cleavage. In another example, other genes involved in xcex3-secretase activity, such as the presenilins, may be altered to identify agents that can compensate for these mutations. Specifically, mutations known to occur in the population can be used to identify agents that are particularly useful in the treatment of subjects having such mutations.
In another embodiment, the assay utilizes an agent that increases xcex3-secretase activity, e.g. cardiolipin or other phospholipids, to increase the sensitivity of the assay.
An object of the invention is to identify therapeutic agents that inhibit the activity of xcex3-secretase, thus inhibiting production of Axcex2, and more specifically Axcex242. Such agents are useful to prevent formation of neuritic plaques in subjects in need of such, for example subjects at risk for familial AD.
Yet another object of the invention is the method of isolation of complexes or components comprised of an agent and the molecules involved in xcex3-secretase activity. This can be used to identify the specific molecules involved in xcex3-secretase activity as well as the specific mutations of these molecules. Isolation of these complexes can be accomplished via techniques such as co-immunoprecipitation.
One advantage of the assay of the invention is that it more authentically reflects in vivo changes in xcex3-secretase activity with the native APP substrate compared to other assays using peptide or semi-synthetic substrates. The membranes are also solubilized which aids in the ease of performing the assay and in reconstitution and purification of xcex3-secretase activity.
Another advantage of the assay of the invention is that it allows efficient delivery of potential agents affecting xcex3-secretase activity interaction with the natural enzyme without the problems of whole cell assays or microsomal assays, e.g. penetration of the agent across the cell membrane
Yet another advantage is that the xcex3-secretase-agent complexes produced in the assay of the invention may be more easily purified than the xcex3-secretases themselves, and thus the assay may be useful to determine the actual molecules involved in the proteolysis and/or secretion of the proteolytic products.
Yet another advantage of the assay of the invention is that xcex3-secretase activity is determined by a direct measurement of xcex3-secretase proteolytic products.
Yet another advantage of the assay of the invention is that components, such as phospholipids, can be added which enhance the xcex3-secretase activity and/or stabilize xcex3-secretase products, e.g., Axcex2, p3 and/or xcex3CTF.
Yet another advantage of the assay of the invention is that the assay is cell-free, and yet does not rely on one subcellular organelle as do other assays such as microsomal assays. The described assays are thus more reflective of the xcex3-secretase activity in all parts of the cell, including the endoplasmic reticulum, the golgi, etc.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.